Differential response of the two cotyledons of Vigna radiata in vitro

Summary Cotyledons of mature seeds of Vigna radiata were found to be variable in their response to N 6-benzytadenine, kinet in and zeatin. The two cotyledons of a seed were designated as CE and C; where CE referred to the cotyledon that remained closely attached to the embryonal axis, and the other more loosely attached cotyledon was referred to as C. Shoots formed from the proximal end of both explants in all nine cultivars studied. Shoot regeneration was faster and regeneration efficiency was higher in CE explants than in C explants in these cultivars. BA was found to be the most suitable cytokinin for both multiple shoot induction and regeneration.Abbreviations BA N6- benzyladenine - Cv cultivar - KIN kinetin - NAA naphthalene acetic acid - RE regeneration efficiency - ZEA zeatin     

Cloning and characterization of two catA genes in Acinetobacter lwoffii K24.

Two novel type I catechol 1,2-dioxygenases inducible on aniline media were isolated from Acinetobacter lwoffii K24. Although the two purified enzymes, CD I1 and CD I2, had similar intradiol cleavage activities, they showed different substrate specificities for catechol analogs, physicochemical properties, and amino acid sequences. Two catA genes, catA1 and catA2, encoding by CD I1 and CD I2, respectively, were isolated from the A. lwoffii K24 genomic library by using colony hybridization and PCR. Two DNA fragments containing the catA1 and catA2 genes were located on separate regions of the chromosome. They contained open reading frames encoding 33.4- and 30.4-kDa proteins. The amino acid sequences of the two proteins matched well with previously determined sequences. Interestingly, further analysis of the two DNA fragments revealed the locations of the catB and catC genes as well. Moreover, the DNA fragment containing catA1 had a cluster of genes in the order catB1-catC1-catA1 while the catB2-catA2-catC2 arrangement was found in the catA2 DNA fragment. These results may provide an explanation of the different substrate specificities and physicochemical properties of CD I1 and CD I2.     

Cloning and characterization of two rhamnogalacturonan hydrolase genes from Aspergillus niger.

A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme. In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expression of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently induced after 3 h of growth on apple pectin. The rhamnogalacturonan hydrolase B gene was not induced by apple pectin, but the rhgB gene was derepressed after 18 h of growth on either apple pectin or sucrose. Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain high-producing A. awamori transformants which were then used for the purification of the two A. niger rhamnogalacturonan hydrolases. High-performance anion-exchange chromatography of oligomeric degradation products showed that optimal degradation of an isolated highly branched pectin fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogalacturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeatus rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5). Compared to the A enzymes, the A. niger B enzyme appears to have a different substrate specificity, since additional oligomers are formed.     

Purification and characterization of tubulin from mung bean (Vigna radiata)

Tubulin has been purified from mung bean seedling by Zn²⁺-induced polymerization. Both α- and β-subunits of mung bean tubulin are different from those of brain tubulin in electrophoretic mobility, colchicine binding and peptide map. Heterogeneity of mung bean tubulin has also been documented suggesting diversification of tubulin despite its conserved nature in general.     

Cloning and characterization of two Serratia marcescens genes involved in core lipopolysaccharide biosynthesis.

Bacteriocin 28b from Serratia marcescens binds to Escherichia coli outer membrane proteins OmpA and OmpF and to lipopolysaccharide (LPS) core (J. Enfedaque, S. Ferrer, J. F. Guasch, J. Tomás, and M. Requé, Can. J. Microbiol. 42:19-26, 1996). A cosmid-based genomic library of S. marcescens was introduced into E. coli NM554, and clones were screened for bacteriocin 28b resistance phenotype. One clone conferring resistance to bacteriocin 28b and showing an altered LPS core mobility in polyacrylamide gel electrophoresis was found. Southern blot experiments using DNA fragments containing E. coli rfa genes as probes suggested that the recombinant cosmid contained S. marcescens genes involved in LPS core biosynthesis. Subcloning, isolation of subclones and Tn5tac1 insertion mutants, and sequencing allowed identification of two apparently cotranscribed genes. The deduced amino acid sequence from the upstream gene showed 80% amino acid identity to the KdtA protein from E. coli, suggesting that this gene codes for the 3-deoxy-manno-octulosonic acid transferase of S. marcescens. The downstream gene (kdtX) codes for a protein showing 20% amino acid identity to the Haemophilus influenzae kdtB gene product. The S. marcescens KdtX protein is unrelated to the KdtB protein of E. coli K-12. Expression of the kdtX gene from S. marcescens in E. coli confers resistance to bacteriocin 28b.     

Isolation and characterization of genes encoding lipid transfer proteins in Linum usitatissimum

Very little is known about lipid transfer proteins from flax (Linum usitatissimum L.). In the present work, three genes encoding a lipid transfer protein (LTP) were isolated from flax, two of which encoded Type-1 and one Type-2 LTPs with molecular masses of about 9 and 7 kDa, respectively. The analysis of deduced amino acid sequence reveals that only Type 2 of the L. usitatissimum leaf specific LTP (LuLTP_Ls) had an N terminal signal peptide consisting of 23 amino acids. The phylogenetic analyses of LuLTP_Ls suggest their closest relatedness with respective proteins from Dimocarpus longan and Vitis vinifera. The gene expression analysis shows that LTP Type 1 genes, which include LuLTP_Ls1 and LuLTP_Ls3, were progressively expressed during leaf development, whereas LuLTP_Ls4 (Type 2) was expressed only at initial and terminal senescence stages of cotyledons. The results suggest that both types of LuLTP_Ls were differentially yet significantly expressed in cotyledons implicating their function in transport and scavenging lipidic skeletons for the benefit of other developing parts of the plant.     

Isolation and characterization of lipid transfer protein (LTP) genes from a wheat-rye translocation line

. Two genes (TaLTP1 and TaLTP2) encoding lipid transfer proteins (LTPs) were isolated from a cDNA library constructed from leaf tissue harvested from 4-week-old seedlings of a wheat-rye near-isogenic line (NIL) involving a translocation of rye chromosome 2RL with wheat 2BS. The spatial and temporal patterns of expression of TaLTP1 and TaLTP2 were examined by Northern blot analysis and in situ hybridization. Both TaLTP1 and TaLTP2 contained a 270-bp open reading frame and encoded a putative LTP precursor molecule of 90 amino acids. Expression of the two LTPs was detected in leaves, stems, and crowns of the NILs but not in the roots. The expression levels of TaLTP1 and TaLTP2 remained constant in response to cold and ABA treatments over a period of 24 h but increased 3 days after the initiation of drought stress. An in situ hybridization study indicated that TaLTP1 was expressed in the cells within the vascular bundles of leaves and in the tissue layers between the vascular bundles in the crowns of the control and drought-treated plants. Expression of TaLTP1 in the tissue layers between the vascular bundles was higher in the drought-treated plants than in the control plants. The results suggested that high levels of expression of TaLTPs in the tissue layers between the vascular bundles might play a role in the drought tolerance response of the wheat crown.     

Cloning and characterization of two novel DNases from Streptococcus pyogenes

The proteins in the culture supernatant (exoproteins) from Streptococcus pyogenes serotype M1 were separated by two-dimensional gel electrophoresis, and their N-terminal amino acid sequences were determined. The amino acid sequences were compared to sequences in the S. pyogenes genome database. The coding sequence showed similarity to sequences of two genes, mf2-v ( mf2 variant) and mf3, which had sequence similarity to genes encoding mitogenic factor (MF); MF has DNase activity. The recombinant genes were expressed in Escherichia coli and the proteins were synthesized. Mf2-v and Mf3 had DNase activity. The activity of Mf2-v was localized to the C-terminal half of the protein. The mf3 gene was shown to be present in most clinically isolated strains of S. pyogenes tested, and the mf2gene was detected in 20% of the isolates. The products of the mf2 and mf3 genes in clinically isolated S. pyogenes strains were thus shown to be DNases.     

绿豆种子8S球蛋白基因启动子的克隆及分析

根据绿豆种子8S球蛋白α'亚基基因的末端序列设计3个特异反向引物.以绿豆基因组DNA为模板,采用基因组步移法,获得了8S球蛋白α'亚基基因起始密码子上游784 bp的DNA片段,通过序列测定和生物信息学分析,发现该序列含有启动子核心区以及大量的种子特异表达相关的顺式作用元件,表明此序列为种子特异启动子序列.通过PCR方法在启动子3'端加上翻译增强序列TMV-omega序列,构建了植物双元表达载体pBI-8SG α'-omega-gus,并成功将其转化进入农杆菌.     

Ontogeny of Somatic Embryos of Vigna aconitifolia, Vigna mungo and Vigna radiata

Callus cultures were initiated from immature cotyledons of Vignaaconitifolia, V. mungo and V. radiata on MS medium supplementedwith NAA, picloram or 2, 4-D. On transfer to L-6 liquid mediumsupplemented with low concentrations of picloram, GA₃ and cytokinins,large number of somatic embryos differentiated from the callus.The cells destined to become somatic embryos divided to formspherical or filamentous proembryos. From the filamentous proembryo,the embryo proper developed either at single or multiple sites.Development of somatic embryos from multiple sites resultedin several embryos connected by a common suspensor at the radicleend. Continued divisions of the proembryos led to globular,heart shaped and dicotyledonary stages of somatic embryogenesis.The somatic embryos of V. mungo and V. aconitifolia differentiatedinto tiny plantlets at low frequency (1%) in liquid suspensioncultures supplemented with zeatin, picloram and GA₃. Vigna aconitifolia Jacq, Marechal, mothbean, Vigna mungo L. Hepper, urdbean, Vigna radiata L. Wilczk, mungbean, somatic embryo     

Composition of Bleeding Sap in Vigna radiata

Bleeding sap and nodules from Vigna radiata were analysed for their free amino nitrogen content and amino acid composition at different stages of growth and development. The bleeding sap contained mostly basic amino acids, whereas the nodules contained both acidic and basic amino acids. The amino nitrogen content of the bleeding sap increased during growth and then declined appreciably during fruit development. In contrast, nodule amino nitrogen declined from seedling stage onwards till flowering, increased during fruit development and then declined again. Nitrate reductase activity in the leaves examined at different stages of development increased from seedling stage onwards and was maximum during early fruit-development stage. It declined during pod-filling stage. The study suggests that the amount of nitrogen fixed from the atmosphere is insufficient, so that the plant has to draw upon soil nitrogen as well. This may be necessary due to the high demand of nitrogen during pod filling.     

Cloning and characterization of nuclear genes for two mitochondrial ribosomal proteins in Saccharomyces cerevisiae.

The genes for two large subunit proteins, YmL8 and YmL20, of the mitochondrial ribosome of Saccharomyces cerevisiae were cloned by hybridization with synthetic oligonucleotide mixtures corresponding to their N-terminal amino acid sequences. They were termed MRP-L8 and MRP-L20, respectively, and their nucleotide sequences were determined using a DNA sequencer. The MRP-L8 gene was found to encode a 26.8-kDa protein whose deduced amino acid sequence has a high degree of similarity to ribosomal protein L17 of Escherichia coli. The gene MRP-L20 was found to encode a 22.3-kDa protein with a presequence consisting of 18 amino acid residues. By Southern blot hybridization to the yeast chromosomes separated by field-inversion gel electrophoresis, the MRP-L8 and MRP-L20 genes were located on chromosomes X and XI, respectively. Gene disruption experiments indicate that their products, YmL8 and YmL20 proteins, are essential for the mitochondrial function and the absence of these proteins causes instability of the mitochondrial DNA.     

Cloning and characterization of a novel mannose-binding protein of Acanthamoeba

Acanthamoebae produce a painful, blinding infection of the cornea. The mannose-binding protein (MBP) of Acanthamoeba is thought to play a key role in the pathogenesis of the infection by mediating the adhesion of parasites to the host cells. We describe here the isolation and molecular cloning of Acanthamoeba MBP. The MBP was isolated by chromatography on the mannose affinity gel. Gel filtration experiments revealed that the Acanthamoeba lectin is a approximately 400-kDa protein that is constituted of multiple 130-kDa subunits. Cloning and sequencing experiments indicated that the Acanthamoeba MBP gene is composed of 6 exons and 5 introns that span 3.6 kb of the amoeba genome and that MBP cDNA codes for a precursor protein of 833 amino acids. That the cloned cDNA encodes authentic MBP was demonstrated by showing that: (i). recombinant MBP possesses mannose binding activity, and (ii). polyclonal antibodies prepared against Acanthamoeba MBP bound to the recombinant protein. Sequence analysis revealed that the MBP contains a large N-terminal extracellular domain, a transmembrane domain, and a short C-terminal cytoplasmic domain. Despite extensive BLAST searches using the MBP sequence, no significant matches were retrieved. The most striking feature of the Acanthamoeba MBP sequence is the presence of a cysteine-rich region containing 14 CXCXC motifs within the extracellular domain. In summary, we have isolated, cloned, and characterized a novel MBP from Acanthamoeba. Because the presence of antibodies to MBP in tears provides protection against infection, the availability of the MBP cDNA sequence and rMBP should help develop: (i). a tear-based test to identify individuals who are at risk of developing the keratitis and (ii). strategies to immunize high-risk individuals.     

Crystal structure of Vigna radiata cytokinin-specific binding protein in complex with zeatin

The cytosolic fraction of Vigna radiata contains a 17-kD protein that binds plant hormones from the cytokinin group, such as zeatin. Using recombinant protein and isothermal titration calorimetry as well as fluorescence measurements coupled with ligand displacement, we have reexamined the K(d) values and show them to range from approximately 10(-6) M (for 4PU30) to 10(-4) M (for zeatin) for 1:1 stoichiometry complexes. In addition, we have crystallized this cytokinin-specific binding protein (Vr CSBP) in complex with zeatin and refined the structure to 1.2 A resolution. Structurally, Vr CSBP is similar to plant pathogenesis-related class 10 (PR-10) proteins, despite low sequence identity (<20%). This unusual fold conservation reinforces the notion that classic PR-10 proteins have evolved to bind small-molecule ligands. The fold consists of an antiparallel beta-sheet wrapped around a C-terminal alpha-helix, with two short alpha-helices closing a cavity formed within the protein core. In each of the four independent CSBP molecules, there is a zeatin ligand located deep in the cavity with conserved conformation and protein-ligand interactions. In three cases, an additional zeatin molecule is found in variable orientation but with excellent definition in electron density, which plugs the entrance to the binding pocket, sealing the inner molecule from contact with bulk solvent.     

Cloning and characterization of a novel Ca2+/calmodulin-dependent protein kinase I homologue in Xenopus laevis

In order to investigate protein kinases expressed in the different developmental stages of Xenopus laevis, recently developed expression cloning was carried out. When two different expression libraries, Xenopus oocyte and Xenopus head (embryonic stage 28/30) cDNA libraries, were screened by kinase-specific monoclonal antibodies, cDNA clones for various known and novel protein serine/threonine kinases (Ser/Thr kinases) were isolated. In addition to well-characterized Ser/Thr kinases, one cDNA clone for a putative kinase was isolated from the Xenopus head library. The sequence of the open reading frame of the cDNA encoded a protein of 337 amino acid residues with a predicted molecular weight of 38,404. Since the deduced animo acid sequence of this protein was 75% identical to that of rat Ca(2+)/calmodulin-dependent protein kinase I (CaMKI), it was designated as CaMKIx. Although recombinant CaMKIx expressed in Escherichia coli showed no protein kinase activity against syntide-2, a synthetic peptide substrate, it was activated when phosphorylated by mouse Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaMKKalpha). Activated CaMKIx significantly phosphorylated various proteins including synapsin I, histones, and myelin basic protein. CaMKIx could not be detected in the early stages of embryogenesis, but was detected in late embryos of stages 37/38 and thereafter when examined by Western blotting using a specific antibody. This kinase was found to be highly expressed in adult brain and heart, and an upstream kinase that could activate CaMKIx was detected in these tissues. These results suggest that CaMKIx plays some critical role in the late stages of embryogenesis of Xenopus laevis.